Instrumental quiz 4

chromatography

separation methods that use the fact that analytes dissolved into a mobile phase will interact differently with the stationary phase (sequential solute exchange between immiscible phases)

preparative scale

collecting eluting solvent in fractions

chromatogram

characterizes the sample using a detector over time

liquid-liquid extraction

most basic type (eg aqueous and organic layers)

1. use ICE table to set up equation for KD

2. KD=[a]organic/[a]aqueous

3. solve for x

fraction of moles in aqueous step

q=(volume aqueous)/(volume organic)(KD)+(volume aqueous)

how to increase separation of liquid-liquid extraction

do more extractions with smaller volumes rather than one big one

how does a column work

similar to a repeat liquid-solid extraction, the solids in the stationary phase are like branches in a creek and analyte get stuck on the branches for different amounts of time depending on their structure/characteristics (creek=mobile phase)

tm

time to elute unretained solutes (aka mobile phase going through entire column)

tr

retention time of components based on the KD value (solute equilibrium between stationary and mobile phase)

high KD

more time stuck in the stationary phase, higher tr

ka

retention factor, =(tr-tm)/(tm)

why do peaks have different widths?

multiple paths, low number of N or large H, solute’s movement within the mobile and stationary phases is not in equilibrium (mass transfer)

van Deemter Equation

combines all 3 band broadening factors H=A+(B/u)+C

ideal flow rate

want to make sure that we have enough theoretical plates and small enough plate height for the best separation, but leaving compounds on the column for too long can decrease volume of the eluting stuff

full separation of peaks

resolution between peak A and peak B must be greater or equal to 1.5

Ways to maximize number of plates

• change u, the linear velocity of the mobile phase

• reduce the diameter of packing particles

• reduce mobile phase viscosity or increase column temperature

ways to increase a (selectivity factor)

• adjust the stationary phase to retain A less

• adjust mobile phase composition to solubilize A better

ways to increase kB

• adjust stationary phase to retain B more

• adjust mobile phase composition to solubilize B better

what are the 2 main applications of chromatography

qualitative analysis or quantitative analysis

GC-FAME

Gas chromatography-Fatty acid methyl ester analysis converts lipids in the cell wall to fatty acid esters which are separated and detected by GC, each bacteria has its own lipid profile

esterification

step used to prep bacterial cell walls for GC-FAME, react the carboxylic acid of the lipid with an alcohol to make an ester

steps of GC-FAME

1. culture homogenous populations

2. esterification

3. use silicone column and H2 mobile phase, flame ionization detector

4. compare fatty acid profile to a library of profiles

gas chromatography mobile phase

gas at all operating temperatures, need sample able to vaporize easily (small)

solute elution of gas chromatography

reflects relative vapor pressure/boiling point and molecular interaction between solute and stationary phase

Open Tubular vs. Packed Columns

Open tubular columns have better resolution for GC because they are longer, have higher flow rates, better sensitivity. LC uses packed columns because it has higher sample capacity.

column stationary phase

needs to be made up of nonvolatile/chemically unreactive- can be non-polar, intermediate polarity, and strongly polar

flame ionization detector

burns the sample with a flame, sample loses an electron, electrons create a current. this has good detection limits and is sensitive, but destructive and not universal

optimizing GC

• temperature programming to change elution rate

• changing the stationary phase

• avoiding overloading the capacity

HPLC advantages

separation, no vaporization, easier prep, more modes to separate compounds with

2 main applications of HPLC

preparative: separating large amount of mixture into components

analytical: goal is to characterize mixture (small amount)

normal phase HPLC

less polar mobile phase and polar solid stationary phase

reverse phase HPLC

polar mobile phase solvent and nonpolar solid stationary phase

why are LC plate heights smaller than GC?

Diffusion in liquid is slower than gas, but LC has smaller diameter columns, uniform flow, and low travel distance between particles

isocratic elution

constant mobile-phase composition

gradient elution

elution with varying mobile phase composition

increasing solvent strength

making the mobile phase less polar (or more if reverse)

injection loop

since HPLC must be kept at high pressure, this must be used to control the flow of the sample onto the column without introducing the system to the low pressure of the environment

guard columns

small column placed before analytical column with the same stationary phase that captures contaminants and irreversibly bound solutes (filter for analytical)

Size exclusion chromatography

large molecules elute first because small ones hang out in small cracks in the stationary phase

ion exchange

stationary phase is charged and interacts with ions, binding with ions of the opposite charge (needs to then be washed to remove bound ions), often used for proteins

flow of mass spec

1. analyte molecules are atomized, vaporized, ionized

2. ions accelerated into mass spec/analyzer

3. ions separated by m/z ratio (with electric or magnetic fields)

4. separated ions detected

mass spec measures

frequency of an atom ionization by the mass to charge ratio (m/z)

mass selector

need to separate ion fragments so that they can be detected one at a time, needs to be in vacuum to prevent side reactions

magnetic sectors

based on interactions of charged particles with magnetic fields, velocity is selected for and then mass determines the movement of the ion. can be a single slit or scan the magnetic field with varied current

double focusing magnetic sector

uses one slit to select for velocity and one for spatial detection (better resolution than 1 slit)

time of flight mass analyzer

accelerates ions through a potential and sees how long it takes to get to the end, heavier ions take longer

Reflectron

“ion mirror” electric field that reverses the direction of flying ions in the TOF, increases resolution by compensating for different kinetic energies

quadrupole

4 poles that have different magnetic fields moving through them- low and high m/z rejected and only ions with specific m/z pass through the filter (then scan voltages to change what ions can pass through), fastest but lowest res

applications of each mass analyzer

magnetic sector- highest resolution, like individual isotopes

TOF- polymers and bigger molecules

quadrupole- high speed and lower resolution things

how electron ionization works

• an electron from the source approaches the molecule and hits its electron cloud

• an electron leaves the cloud (very little molecular ion made)

chemical ionization

reagent gas is ionized and then ionizes the sample

MALDI (Matrix-assisted laser desorption/ionization)

laser hits analyte in the matrix on a metal plate, they fly off and are ionized into a plume

Electrospray ionization

liquid analyte sprayed out of needle that has an electric potential, releases a charged spray of fine droplets that enter capillary- mostly molecular ion fragment

Desorption Electrospray Ionization (DESI)

charged tip sprays out particles which then interact with the analyte, making them fly off

inductively coupled plasma (ICP)

ionizes elements (hard source because it causes fragments)

application of chemical ionization

gasoline fragments or volatile things

MALDI or DESI

insulin, other large protein molecules

Electrospray ionization

acetaminophen, other pharma that might use LC

ICP

heavy metals in water, any elemental MS

MS chemical imaging

can use MALDI-TOF mass spec to get mass spectrums of different parts of the sample surface, showing molecule distributions

Ion transducer

ms detector that works like a photomultiplier tube (magnifies input e-), ion impact dislodges one e- that accelerates to point with higher V and impact releases more e- (KE impacts response, more sensitive to smaller mass)

base peak

most abundant peak in spectrum, assigned abundance of 100%

molecular ion/parent peak

molecule minus one electron

nitrogen rule

m/z value of a molecular ion is always an even number if the ion has an even number of nitrogen atoms

M+1 peak

number of carbon atoms in the molecule= (M+1)/(M)*(100/1.1), then round

M+2 peak

indicates presence of chlorine (if 1/3 of M peak), bromine (if about the same of M peak), or sulfur

Rings and Double Bonds Equation

number of rings/double bonds= #c-1/2#h+1/2#N or P + 1

what is likely to ionize first

double bonds or lone pairs